Peptides are small polymers made up of amino acids that have various biological activities. Peptide purification is achieved through chromatography, with HPLC being the most common method. Peptide synthesis is often used to prepare rare peptides, and peptide companies can make custom peptides. Peptide purification is also used to identify proteins and to study novel peptides. Peptides are genetically modified to facilitate purification, and affinity chromatography is used to purify the peptide.
Peptides are small polymers composed of amino acids. Many are biologically active as hormones, toxins or have other capabilities. Such compounds are frequently used in biological, medical and chemical research. They also serve as the building blocks of proteins when a number of them are linked together. Peptide purification is a technique used to purify large quantities of a desired peptide or to help separate peptides generated by protein digestion.
Peptide purification is accomplished using a technique of chromatography, a way to separate compounds that bind differentially to a physical matrix. High pressure liquid chromatography (HPLC) is commonly used for purification of peptides. The peptide or peptides are applied in a time-changing solvent mixture as they are pumped through a column of small beads under high pressure. Different peptides elute at different times and are detected by a monitor, often a UV spectrophotometer.
When researching peptides, often the substance of interest is rare and cannot be purchased from chemical companies. If the structure of the desired peptide is known, it is often easier to chemically prepare it by peptide synthesis than to isolate the compound from natural sources. Isolating natural products is notoriously difficult. Synthetic peptides are generally purified by HPLC.
Such chemical synthesis can be daunting to an independent researcher who is not a chemist. Often, this task is outsourced to peptide synthesis companies that specialize in these techniques. This can be cheaper than trying to set up the system from scratch in a lab. Peptide companies can make custom peptides tailored to the researcher’s needs.
Another reason for peptide purification can be when a researcher has purified a protein and is trying to determine its identity. He or she can degrade the protein into peptide fragments, separate the fragments by purification, and have the fragmentation pattern of the peptides detected by a mass spectrometer as they elute from the column. This technique is known as LC-MS, short for liquid chromatography-mass spectrometry. It provides the molecular weight and amino acid composition of the fragments, and often allows for the identification of proteins, if similar or identical ones have previously been identified.
Many researchers work with peptides that have novel characteristics, such as unnatural amino acids, to try to find ones with unusual biological activities. There is an entire field called peptidomimetics dedicated to studying such new peptides. Often computer-generated sequences are designed and a peptide library is synthesized comprising a range of atypical peptides. Peptide purification is used to separate individual members of this library to provide pure peptide for testing biological activity. This strategy has been successful in generating at least one new commercially available drug.
Many of the biologically active peptides are of interest for medical uses. Commercially used compounds, such as insulin, are commonly produced by recombinant DNA overexpression systems that generate large quantities of the desired compound. Often, peptides are genetically modified to facilitate purification by adding some sort of tag to their fronts. This allows for the use of affinity chromatography to purify the peptide.
With this type of chromatography, the tag is a compound, such as histidine, that will bind an array of beads, such as nickel, chosen for its ability to bind the tag. Unwanted proteins and peptides generally pass through the column without binding. The specially designed peptide usually binds strongly to the column. After the contaminating proteins and peptides have been washed away, the desired peptide is eluted from a compound that competes for binding to the matrix. This results in a fairly pure preparation of the desired peptide and the tag can be removed by cleavage.
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